A novel genotype of avian hepatitis E virus identified in chickens and common pheasants (Phasianus colchicus), extending its host range

In 2019, outbreaks of hepatitis-splenomegaly syndrome (HSS) were observed in six commercial layer chicken flocks, belonging to three different Polish farms, and characterized by increased mortality, hemorrhagic hepatitis with attached blood clots on the liver surface, and splenomegaly. Diseased flocks were initially investigated for the presence of avian hepatitis E virus (aHEV) – the etiological agent of HSS – by conventional reverse transcriptase polymerase chain reaction, which revealed aHEV sequences clustering separately from all known aHEV genotypes. Additionally, an aHEV genome was identified for the first time in common pheasants, from a flock in France, using Next Generation Sequencing. This genome clustered together with the Polish aHEVs here investigated. Complete genome aHEV sequences from the HSS outbreaks confirmed the divergent cluster, with a shared nucleotide sequence identity of 79.6–83.2% with other aHEVs, which we propose to comprise a novel aHEV genotype – genotype 7. Histology and immunohistochemistry investigations in the liver and spleen established an association between aHEV and the observed lesions in the affected birds, consolidating the knowledge on the pathogenesis of aHEV, which is still largely unknown. Thus, the present investigation extends the natural host range and genotypes of aHEV and strengthens knowledge on the pathogenesis of HSS.

www.nature.com/scientificreports/ and pathogenesis. Thus, epidemiological studies with a focus on genotyping and host tropism are important to fulfill such gaps in knowledge.
In the present study, we report nearly complete genome sequences and phylogenetic analysis of aHEVs belonging to a novel genotype, which were detected in various outbreaks of HSS in layer chicken flocks in Poland, but also in pheasants from France.

Methods
Outbreak description and field investigations. In 2019, several commercial layer chicken flocks in Poland, belonging to three different multi-age poultry farms-A, B, and C (Table 1)-experienced increased mortality. During post-mortem investigations, samples of liver, spleen, and bile of diseased chickens were collected in FTA® cards and shipped for further laboratory investigations. In three flocks, cloacal swabs were also collected from affected birds (Table 1).
In a distinct case, commercial pheasant flocks from France suffered from outbreaks of hepatitis with high mortality. The description of the outbreaks and performed investigations, including collected samples and subsequent analyses, have been recently reported elsewhere 16 . www.nature.com/scientificreports/ Histopathology. Liver and spleen samples were processed for histology, being initially fixed in a 4% neutral buffered formaldehyde solution (SAV LP GmbH, Flintsbach, Germany), followed by a dehydration procedure and paraffin embedding. The formalin-fixed paraffin-embedded (FFPE) samples were then cut into sections of 4 µm with a microtome (Microm HM 360; Microm Laborgerate GmbH, Walldorf, Germany), mounted on glass slides, and stained with hematoxylin and eosin (H&E) for microscopic assessment.
Immunohistochemistry for the detection of aHEV. A truncated recombinant aHEV capsid protein, designated ORF2-1, was expressed as previously reported 17 and inoculated in rabbits to produce polyclonal antibodies. For immunohistochemistry (IHC) detection of aHEV in tissues, additional sections of FFPE liver and spleen samples were obtained (4 µm) by a microtome (Microm HM 360) and mounted on positively charged glass slides (Superfrost plus; Menzel-Gläser, Braunschweig, Germany). Liver and spleen samples of specificpathogen-free chickens, both healthy birds and those suffering from hepatitis (due to fowl adenovirus infection), were included to study the specificity of the polyclonal serum. After the first step of dewaxing and rehydration, the slides were heated in citrate buffer (pH 6.0), for antigen retrieval. The activity of endogenous peroxidase was then blocked with 1.5% H 2 O 2 in methanol, for 30 min. As a blocking step, the sections were incubated with a 1:10 dilution of normal goat serum (Vector Laboratories, Burlingame, USA) mixed with 2% bovine serum albumin (Roche Diagnostics GmbH, Mannheim, Germany) for 60 min, at room temperature, in a humidified chamber. Next, the slides were incubated overnight, at 4 °C, with the primary antibody (purified rabbit polyclonal anti-ORF2 aHEV serum) at three dilutions: 1:500, 1:1000, and 1:1500. As a control for the primary antibody, additional sections were incubated with PBS. The sections were then extensively washed in PBS, and incubated with a 1:400 dilution of biotinylated anti-rabbit IgG (Vector Laboratories) for 30  Next generation sequencing. , which consisted of FFPE liver and bursa of Fabricius, derived from a pheasant flock in France, suffering from hepatitis and high mortality, was investigated by deep sequencing using the Illumina NextSeq platform, as it was comprehensively described elsewhere 16 . To extract only contigs with aHEV specific sequences, all viral contigs with size ≥ 100 bp were compared against a database set up to contain all available Orthohepevirus B sequences, using the BLASTn algorithm. Based on the best BLASTn-scores (E-value ≤ 10 -10 ), metagenomics contigs of up to near-complete genome length of an aHEV strain were identified.
Obtaining near-complete genome sequences. Based on the aHEV sequence identified in the pheasant sample, eight primer pairs covering the nearly complete genome were designed and applied to chicken samples ( Table 2). All PCRs to obtain the whole aHEV genomes of samples 19-13931 and 19-27337 were performed with the OneStep RT-PCR kit (Qiagen) and 15 pmol of each primer ( Table 2). The thermal profile was as follows: 30 min at 50 °C for reverse transcription, 15 min at 95 °C for initial PCR activation and denaturation, followed by 40 cycles of 30 s at 94 °C, annealing temperature as in Table 2

Results
Clinical features of the HSS outbreaks. Mortality in selected flocks A1, A3, and C reached the highest weekly values of 2%, 1.2%, and 1.7%, four weeks after the onset of increased mortality, with cumulative values of 6.7%, 5%, and 9%, respectively, being recorded after six weeks (Fig. 1a). The affected flocks were between 27 and 68 weeks of age and were kept in a cage system. Post-mortem investigations revealed friable, swollen livers, with hepatitis, subcapsular hematomas, and /or attached blood clots on the surface, and splenomegaly (Fig. 1b, c).
Histopathology analysis. The histology assessment of liver samples revealed multifocal areas of coagulative necrosis or generalized necrosis, infiltrations of heterophils and mononuclear cells, and several areas of hemorrhages. Additionally, sinusoidal deposits of eosinophilic, amorphous, hyaline material, suggestive of amyloid, were observed, which disrupted the hepatic plates (Fig. 2a). Some liver samples also presented granulomas and colonies of rod-shaped bacteria. Likewise, the analysis of spleen samples revealed multifocal to generalized areas of necrosis, infiltration of heterophils, and hemorrhages. In addition, severe and extensive disruption of the splenic tissue by homogenous, amorphous, eosinophilic, proteinaceous material consistent with amyloid, was a common finding (Fig. 2b).
Positive signals for aHEV ORF2 were detected by IHC in both liver and spleen samples of affected chickens. In the liver, positive signals were observed in the hepatic sinusoids and in accumulations of mononuclear cells (Fig. 2c, d), while in the spleen positive signals were more present in the peri-ellipsoidal zones, throughout the organ (Fig. 2e). The positive signals were more distinct and with the least background with a primary antibody dilution of 1:1000. No positive signals were observed in the controls investigated.
Discovery of a novel aHEV genotype. Initial de novo metagenome assembly of sample 19-03914 from pheasants yielded 67,302,080 reads, which were assembled into 3881 contigs of lengths varying from 362 to 223,074 nucleotides. The obtained metagenomics profiling identified different families of bacteria and viruses. Furthermore, the viral contigs were classified into five different categories with the largest portion (86%) corresponding to double-stranded (ds) DNA viruses (Fig. 3). Among the others, a genome of aHEV was identified, and the presence of aHEV RNA in the sample 19-03914 was, then, confirmed by RT-qPCR, according to a previously published protocol 19 . The assembled sequence of 6639 nucleotides in length covered the near-complete aHEV genome, excluding only the terminal portions of the 5' and 3' non-coding regions (NCR). The complete genome sequence was submitted to the NCBI database under the accession number ON922634.
The phylogenetic analysis was based on whole genome sequences belonging to the species Orthohepevirus B. All aHEV genomes reported here clustered together in a separate branch, representing a new putative aHEV genotype (Fig. 4), with a shared nucleotide sequence identity of 98-99.3%, and a 79.7-83.3% nucleotide identity with other aHEVs (Supplementary Table S1-Percent identity complete genomes).  www.nature.com/scientificreports/

Discussion
Since its first clinical appearance in 1988 in Australia, serological and molecular studies have revealed a worldwide prevalence of aHEVs in chicken flocks, which however does not translate necessarily in a clinical condition 5 . Additionally, histopathological lesions produced by aHEV are etiologically unspecific 2 . The impressive liver lesions noticed macroscopically can easily be confounded with amyloidosis, as described in context with the application of bacterial oil-emulsion vaccines 20,21 . Therefore, in the present study, we established an IHC protocol, with a primary polyclonal rabbit anti-aHEV ORF2 antibody, to confirm the presence of aHEV antigen in organs presenting pathological changes. Our investigations revealed positive signals in RT-PCR-positive samples of liver and spleen from chickens suffering from HSS, establishing an association between aHEV and the observed lesions. The distribution of the positive signals in these organs reinforced the findings of previous studies 22,23 , with aHEV antigen being detected mainly in the hepatic sinusoids in the liver, while in the spleen aHEV antigen was not confined to a particular area. Such findings are important to consolidate the knowledge on the pathogenesis of aHEV, which is still largely unknown. It is established that viral replication initially takes place in gastrointestinal tissues before reaching the liver 5 , and our results seem to further indicate that sinusoidal cells in the liver might play a significant role in the aHEV replication. Additionally, the scattered pattern of antigen observed in the spleen, leading to splenomegaly, suggests the involvement of mononuclear cells in the replication of aHEV.
Since the knowledge on aHEV pathogenesis and aHEV strains' pathogenicity is limited by the lack of an appropriate lab propagation system 5 , it is important to monitor field aHEVs circulating in the bird populations and record associated outbreaks. More recently, a novel genotype identified in China was associated with a more severe form of the disease in chickens denominated by hepatic rupture hemorrhage syndrome (HRHS), leading to a cumulative mortality of 15% in the affected flocks 24 . Similarly, the HSS outbreaks reported in the present study lead to high mortality rates of up to 2% per week, and up to 9% cumulatively. More detailed epidemiological investigations would be needed, including clinically healthy birds, to correlate between the genotype and pathogenicity of a certain aHEV strain.
After being identified as the causative agent of the BLS disease/HSS in chickens 25,26 , genetic characterization studies revealed, throughout the years, heterogeneity of aHEV field strains 6,13,27,28 . In this regard, four distinct aHEV genotypes were proposed initially, which at that time correlated to the different geographical locations where the virus was identified 4 . More recently, two additional genotypes have been proposed from the analysis of the complete genome of aHEV strains identified in China 14,15 . In the present investigation, we report nearly complete aHEV genomes identified in pheasants and layer chickens, which belong to a novel aHEV genotype-genotype 7.
Initially, we identified the new aHEV genome by NGS in organ samples of pheasants from France suffering from hepatitis, caused by a novel Chaphamaparvovirus 16 . Since the presence of aHEV RNA was only circumscribed to one pheasant-rearing farm out of 15 investigated, the finding was considered accidental and negligible to the clinical outcome as it was undoubtedly attributed to the parvovirus. Additionally, it is well known that birds can be PCR-positive for aHEV in the absence of associated disease 5 . Nevertheless, this is the first time that aHEV is reported in common pheasants (Phasianus colchicus), extending its natural host range. Such finding,  www.nature.com/scientificreports/ however, is not unexpected as in the last 6 years different studies identified aHEVs in birds other than chickens, demonstrating a much broader natural host range for aHEV than previously considered 10,11,29,30 . Differently from the findings in pheasants, six layer chicken flocks, with ages between 27 and 68 weeks, belonging to three different farms in Poland, presented a clinical picture compatible with classically described HSS, which included increased mortality, hemorrhagic hepatitis with attached blood clots on the liver surface, and splenomegaly. Diseased flocks were initially investigated for the presence of aHEV by conventional RT-PCR targeting helicase and capsid genes, with the former presenting higher positivity rates than the latter. This is likely because the viral RNA coding for ORF1 (helicase) and ORF2 (capsid) are replicated in different amounts, depending on the stage of infection, with high viral load samples being positive in both RT-PCRs 31 .
Preliminary phylogenetic analyses of partial helicase and capsid sequences obtained from RT-PCR products suggested that the aHEVs identified in the Polish chicken flocks, together with the 19-03914 aHEV genome from pheasants, compose a separate cluster within all known aHEV genotypes. Complete genome aHEV sequences from the Polish cases were obtained to confirm the divergent cluster, which we propose to comprise a novel aHEV genotype. The comparative analysis of all available genomes of Orthohepevirus B species identified genome identity values in a range from 77.7 to 100%, and a 15-20% variation in the complete nucleotide sequences between aHEV genotypes, criteria that the aHEV complete nucleotide sequences here reported fulfil.
Previously, aHEV genotypes 2, 3, and 4 were identified in birds in Poland, revealing that aHEV is widely spread in Polish chicken flocks 11,[31][32][33] . Interestingly, some aHEV partial helicase sequences recently reported from broilers and laying hens in Poland, which were assigned to genotype 4 11 , seem to be phylogenetically related to the aHEV genomes reported in the present study. Applying the BLAST search algorithm with newly identified genomes, recognized the above-mentioned genotype 4 isolates as sequences with high nucleotide identity (98.2-99.7%). This would imply that the novel aHEV genotype reported here is prevalent in Polish flocks; however, to verify such hypothesis analyses using complete genomes from such cases would be necessary.
In conclusion, here we report nearly complete genomes belonging to a novel aHEV genotype, which were identified in chickens, but also for the first time in pheasants, extending the natural host range of aHEVs. In chickens, the outbreaks were characterized by high mortality, and anatomic-pathological lesions compatible with HSS, and the association of aHEV in the clinical picture was confirmed by IHC in the liver and spleen of affected birds. Hence, the present report extends the host range and genotypes of aHEV, consolidating knowledge on the pathogenesis of HSS, and contemplates important questions on aHEV pathogenesis that shall be considered in future studies.

Data availability
The dataset generated and analyzed during the current study is available in the GenBank repository. The sequences are deposited under the following accession numbers: ON922632, ON922633, and ON922634. Additional data that support the findings of this study are available from the corresponding author upon reasonable request.